The demography of Alzheimer's disease is becoming progressively better understood. It is estimated that over 5% of the U.S. population over 65 and over 15% of the U.S. population over 85 are beset with this disease (Cross, A. J., Eur J Pharmacol (1982) 82:77-80; Terry, R. D. et al., Ann Neurol (1983) 14:497-506). It is believed that the principal cause for confinement of the elderly in long term care facilities is due to this disease, and approximately 65% of those dying in skilled nursing facilities suffer from it.
To confound the problem that therapy is at present a matter of experimentation, diagnosis is also unreliable. There is no straightforward diagnostic test, and diagnosis is made by a series of evaluations based on negative results for alternative explanations for the symptomologies exhibited. Assuming that the presence of the disease can be assessed accurately after death by autopsies of the brain, current results show that present diagnostic methods among living individuals carry an approximately 20% rate of false positives.
It would be extremely helpful in effecting appropriate care for patients and in developing therapies to have a straightforward assay method for diagnosing the presence of Alzheimer's disease. The invention described below provides an approach to this diagnosis.
Certain facts about the biochemical and metabolic phenomena associated with the presence of Alzheimer's disease are known. Two morphological and histopathological changes noted in Alzheimer's disease brains are neurofibrillary tangles (NFT) and amyloid deposits. Intraneuronal neurofibrillary tangles are present in other degenerative diseases as well, but the presence of amyloid deposits both in the interneuronal spaces (neuritic plaques) and in the surrounding microvasculature (vascular plaques) seems to be characteristic of Alzheimer's. Of these, the neuritic plaques seem to be the most characteristic (Price, D. L. et al., Drug Development Research (1985) 5:59-68).
The protein which makes up the bulk of these plaques has been partially purified and sequenced. Plaque-rich brains of deceased Alzheimer's patients have been used as a source to extract an approximately 4.2 kd "core" polypeptide, amyloid plaque core protein (APCP), herein referred to as "beta-amyloid core protein." This peptide was designated B-protein by Glenner, G., et al., [Biochem Biophys Res Commun (1984) 120:885-890]. The amino acid sequence of the amino-terminus has been determined [Glenner, G., et al., Biochem Biophys Res Commun (1984) 122:1131-1135; Masters, C. L., et al., Proc Natl Acad Sci USA (1985) 82:4245-4259]. The amino acid sequences reported by the two groups above are identical except that Glenner et al., report a glutamine at position 11 for Alzheimer Disease cerebral vascular amyloid whereas Masters et al., report glutamic acid at position 11. Also, the former authors report that the cerebral vascular amyloid has a unique amino-terminus while the latter authors report that the form found in amyloid plaque cores has a "ragged" amino-terminus--i.e., peptides isolated from this source appear to be missing 3, 7, or 8 amino acids from the amino-terminus. Both groups have shown that the same peptide is found in the amyloid plaque cores and vascular amyloid of adult Downes syndrome-afflicted individuals and report glutamic acid at position 11.
Further studies on the beta-amyloid core protein were also conducted by Roher, A. et al., Proc Natl Acad Sci USA (1986) 83:2662-2666 which showed the complete amino acid composition of the protein, and verified that it matched that of no known protein. The compositions those of Allsop, D., et al., Brain Res (1983) 259:348-352; nor were they in agreement with those published by Glenner or Masters (supra).
Wong, C. W. et al., Proc Natl Acad Sci USA (1985) 82:8729-8732 showed that a synthetic peptide which was homologous to the first ten amino acids of the beta-amyloid core protein described by Masters (supra) was able to raise antibodies in mice and that these antibodies could be used to stain not only amyloid-laden cerebral vessels, but neuritic plaques as well. These results were confirmed by Allsop, D. et al., Neuroscience Letters (1986) 68:252-256 using monoclonal antibodies directed against a synthetic peptide corresponding to amino acids 8-17. Thus, in general, the plaque protein found in various locations of the brain of Alzheimer's patients appears to be similar in immunoreactivity. It is highly insoluble, as shown by the inability to achieve solubilization in many commonly used denaturants such as detergents and chaotropic agents (Masters, supra, Allsop, D., et al., (supra)).
It is believed, by analogy to other amyloid proteins, that beta-amyloid core protein may be formed from a precursor in the peripheral circulatory system or lymphatic system. There are six known instances of disease-associated amyloid deposits in which the nature of the precursor protein for the amyloid protein is known: for primary amyloidosis, the source is an immunoglobulin light chain; for secondary amyloidosis, the precursor is amyloid A protein; for familial amyloid polyneuropathy and senile cardiac amyloidosis, prealbumin or a variant thereof; for medullary carcinoma of thyroid, a procalcitonin fragment; and for hereditary cerebral hemorrhage, gamma-trace fragment (See, e.g., Glenner, G. New England Journal of Medicine (1980) 302:1283; Sletton, K. et al., Biochem J (1981) 195:561; Benditt, et al., FEBS Lett (1971) 19:169; Sletton, K., et al., Eur J Biochem (1974) 41:117; Sletton, K., et al., J Exp Med (1976) 143:993). The foregoing is a partial list and there are at least a number of additional references with regard to procalcitonin fragment as a precursor for the amyloid of the thyroid carcinoma. Alternatively, or additionally, such a precursor for beta-amyloid core protein may be produced in the brain.
It has been described that a protein containing the beta-amyloid core protein sequence within the framework of a larger protein exists (Kang, J et al., Nature (1987) 325:733-736). This protein, which is a potential precursor in vivo to the beta-amyloid core protein, was predicted from the sequence of a cDNA clone isolated from a human fetal brain tissue cDNA library and consists of 695 amino acid residues wherein the amino terminus of the beta-amyloid core protein begins at position 597. By analogy to the above described series, it may be that such a precursor or a fragment thereof circulates in the serum at a level differentiable in Alzheimer's victims relative to unafflicted individuals. Alternatively or additionally, such differences may be detected in the cerebral spinal fluid.
An alternative mechanism which could lead to the production of a beta-amyloid core protein in vivo is suggested by the observation that the sequences encoding the first amino acid (Asp) of the beta-amyloid core protein is directly proceeded in the genome by the codon for a methionine, which is the initiating amino acid for protein synthesis. Selection of this methionine by the translational apparatus of a cell as an initiator methionine, followed by its enzymatic removal by an aminopeptidase as frequently occurs in vivo, would give rise to a protein with the amino terminus of the beta-amyloid core protein.